Hirschsprung disease (HSCR), or aganglionic megacolon, is a relatively common, multifactorial birth defect associated with the lack of intrinsic ganglion cells in the myenteric (Auerbach) and submucosal (Meissner) plexuses in the gastrointestinal tract. Clinically, the symptoms range from mild to severe and involve aganglionosis of the transverse colon and beyond (long segment: L-HSCR), the splenic flexure and the descending colon (short segment: S-HSCR), or the sigmoid colon only (classic HSCR). Research supported by this grant has led to the identification and biology of five genes involved in HSCR susceptibility: the receptor tyrosine kinase RET, RET's ligand glial cell-derived neurotrophic factor (GDNF), the endothelin receptor B (EDNRB), EDNRB's ligand endothelin-3 (EDN3) and the transcription factor SOX10. Others have identified rare mutations in the alternative RET ligand neurturin (NTN), ECE1 (endothelin converting enzyme-1) and the SMAD interacting protein 1 (SMADIP1). In the previous funding period we have demonstrated multigenic inheritance in all forms of HSCR; RET mutations are necessary but not sufficient in all forms of HSCR; the existence of a common non-coding RET mutation; tissue-specific genetic interaction between RET and EDNRB; the location of novel major susceptibility factors; and, a likely interaction between RET and SOD1 (superoxide dismutase 1) as an explanation of HSCR in trisomy 21 patients. We postulate that HSCR is oligogenic, always requiring RET and other interacting disease susceptibility alleles with phenotypic expression depending on the pathways compromised. Proving this hypothesis by identifying the major genes and their specific mutations, and identifying the molecular interactions, forms the basis of this proposal. [unreadable] [unreadable] We focus on using human disease families, mouse models of HSCR gene mutations and functional analyses of mutations to prove our hypotheses. Importantly, sequence-based genomic analysis plays a major role in gene discovery in this project. The long-term objective of this study is to understand the molecular genetic basis of HSCR. More generally, our aim is to develop a paradigm for sequence-based biology in complex, human diseases. [unreadable] [unreadable]